1. Field of the Invention
The present invention relates to a new immunoassay process for cortisol involving neither extraction nor chromatography, based on the surprising properties of certain antibodies directed against cortisol.
The invention provides in particular the antibodies themselves, a process for obtaining them, assay processes for cortisol based on these antibodies, and kits of reagents allowing the use of said processes.
2. Description of the Background Art
Cortisol, the principal glucocorticoid hormone, is synthesized in the fascicular and reticular zones of the adrenal cortex. It is derived from cholesterol under the influence of several enzymes. Its rate of secretion is under hypothalamic-pituitary control. CRF (Corticotropin Releasing Factor) activates the anterior pituitary which produces ACTH, which in turn stimulates the adrenal cortex which synthesizes corticoids, of which 90% is cortisol. In the plasma, the majority of the cortisol circulates in the form bound to plasma proteins: mainly to corticosteroid binding globulin, to albumin and to testosterone estradiol binding globulin; a very small proportion exists in the free form.
A functional exploration of the hypothalamus-pituitary-adrenal cortex axis is largely based on the determination of cortisol. The measurement of cortisol in serum is used essentially during dynamic tests. Free urinary cortisol exhibits a good correlation with the rate of production of the hormone and remains the best hormonal index in cases of hyperadrenocorticism.
The direct determination of cortisol in serum is facilitated by its generally high level in comparison with all the other steroids, with the exception of dehydro-epiandrosterone (DHEA) sulphate which belongs to the family of androgens with a very different structure and is hardly recognized at all by anti-cortisol antibodies. All the radioimmunoassay or enzyme-immunoassay kits on the market propose techniques for the direct measurement of cortisol in sera. They are often of the desired quality (see Table 1 below).
On the other hand, the direct determination of urinary cortisol is a more difficult operation due essentially to the accumulation of metabolites, of which little is often known about their structure and concentration. Until now, the most reliable assay processes for cortisol in this biological medium include an extraction stage with dichloromethane followed by chromatography which ensures specificity. These operations are delicate, long and expensive.
Numerous attempts have been made to produce monoclonal and polyclonal anti-cortisol antibodies, particularly from a cortisol-21-hemisuccinate derivative and above all from a cortisol-3-CMO derivative, without any description in this latter case of specific properties associated with the syn/anti geometrical isomerism of the CMO group. Such antibodies do not permit a correct determination of urinary cortisol: they overestimate the concentrations of cortisol in the urine compared with the results obtained by determination after chromatography.
Three manufacturers: Kodak, Orion and DSL propose the direct determination of urinary cortisol. However, as Table 2 below shows, these processes give very different results from those obtained by determination after extraction and chromatography of the sample.
It would be desirable, therefore, to have a rapid method for the direct measurement of urinary cortisol which gives a result similar to the one obtained by chromatography, a method which is too cumbersome for routine use.